Wheat cultivation began somewhere in the middle east and has been ongoing for perhaps as long as 10,000 years. Today, wheat, rice and maize form the foundation of the human diet in every corner of the world. Of these three, wheat is the most widely grown and is the single greatest source of dietary protein in the human diet.
The importance of wheat and the other cereals, both because they are favorites for human consumption and because of their economic value, have led to their prominence in visions of the future of biotechnology. The full impact of biotechnology on agriculture will occur only when all the necessary technologies are available to cereal scientists, a prerequisite that is yet to be fulfilled. There are many aspects to biotechnological approaches to wheat modification, but the technical requirements can be divided into two basic categories. The first is transformation; i.e., there must be protocols to introduce exogenous genes directly into the wheat genome. Once this DNA is integrated into the wheat genome, it must be stable and passed on to the subsequent generations. Without this technology, the full power of molecular biology cannot be applied to this critical crop. The transformation protocols must be efficient and accessible to many laboratories. In spite of wheat's importance, research and development must balance the significance of the crop under study with the technical ease of manipulation. The second requirement for bioengineering wheat is the possession of genes that researchers have a reasonable expectation will confer positive traits if these genes can be transformed into wheat. Such genes will include those affecting herbicide resistance, insect and viral resistance, storage proteins, and starch metabolism.
Many of the recent advances in plant science have resulted from application of the analytical power of recombinant DNA technology coupled with plant transformation. These approaches facilitate studies of the effects of specific gene alterations on plant development and physiology. They also make possible the direct manipulation of genes to bioengineer improved plant varieties. Although wheat (Triticum aestivum) is the largest crop in the world in terms of production, it was the last among economically important cereals to be transformed. Transformation of monocotyledonous plants, including cereals, has proven much more difficult than dicotyledonous plants, such as tobacco. No proof of transgenic cereal plants has been shown using viral vectors, incubation of dry seeds or embryos in DNA, liposome fusion with tissues and protoplasts, liposome injection, microinjection or electroporation. Vasil et al. (Bio/Technology 9:743-747, 1991) produced stably transformed wheat suspension-cell cultures from which they were unable to regenerate plants. Later, Vasil et al. (Bio/Technology 10:667-674, 1992) obtained several transformed callus lines after microprojectile bombardment of embryogenic callus and selection with the herbicide Basta. Transformed wheat plants were regenerated from one of these lines. These plants were unable to self-fertilize, but progeny could be produced by outcrossing to either wild-type pollen or ova. Although the work of Vasil and collaborators was a landmark in efforts to develop wheat transformation, the protocol they reported was limited in its utility by its dependence on the identification and establishment of a specific callus type in long-term tissue cultures.
A rapid and efficient method of producing transgenic wheat lines has been achieved (Weeks et al., Plant Physiol. 102:1077-1084, 1993). Callus tissue produced by immature embryos shortly after excision and culturing is the target for microprojectile-mediated DNA delivery. The callus tissue can be maintained and regenerated into fertile plants with high frequency. This procedure makes it feasible for the first time to do promoter and protein expression studies. In addition it allows for the introduction of economically and agronomically important traits into wheat, such as disease resistance, insect resistance and seed quality.
Due to the often low transformation frequencies achieved in plants, especially wheat, selectable markers are very important in identifying the transformants. The most commonly used selectable markers are antibiotic resistance genes and herbicide resistance genes. These markers have several disadvantages. The presence and constitutive expression of antibiotic resistance genes, while critical for the selection of transformants, are of no benefit to the plant. The use of antibiotics for selection also raises questions of regulatory approval and public acceptance in addition to problems with natural resistance. Similar problems may be encountered with the use of herbicides. An additional disadvantage of using herbicide resistance genes as selectable markers is the risk of the gene being introduced into the target weeds by natural crosses between the weeds and crops. This is especially critical in sorghum and oats, which can naturally cross with weeds such as Johnson grass and wild oat, respectively. The commercial application of agricultural biotechnology relies on the discovery of selectable markers which do not have such disadvantages. Additionally, selection methods based on the use of two selectable markers on the same tissue require that more selectable markers be found.
A gene from the soil fungus Myrothecium verrucaria, the cah gene, coding for the enzyme cyanamide hydratase, has been isolated and characterized (Maier-Greiner et al., Proc. Natl. Acad. Sci. USA 88:4260-4264, 1991). This enzyme converts cyanamide into urea by catalyzing the addition of a water molecule. Cyanamide is used as a fertilizer in aqueous solution or in the form of its calcium salt. Calcium cyanamide as a fertilizer has the advantage that the product itself and its metabolites do not cause any environmental concern (Rieder, World Crops, January 1981, pp17-20). FIG. 1 shows the breakdown of calcium cyanamide in the soil. Calcium cyanamide has the following advantages over urea or ammonium nitrate-based fertilizers:
1. Calcium cyanamide nitrogen is released gradually, so the amount of nitrogen available to the plant roots is never in excess of plant needs. PA0 2. Calcium cyanamide nitrogen provides a more efficient plant food because losses by leaching, run-off or dentrification are minimized. PA0 3. Fertilizer application costs are lower through reduction in frequency of application. PA0 4. There is no nitrate pollution of ground water, streams and lakes.
Calcium cyanamide also functions as a herbicide due to its metabolite cyanamide, which controls germinating weed seeds and weed seedlings. The use of calcium cyanamide has diminished over the years in favor of hormonetype weed killers, soil herbicides and other specific compounds, which can achieve a 100% weed kill. However, there is increasing concern about the impact such herbicides have on the environment. Calcium cyanamide is effective when weed infestation is low, and when used as a fertilizer, can reduce the need for specific herbicides. Another useful function of cyanamide is as a fungicide, especially for control of eye spot disease in wheat. Calcium cyanamide does not have the disadvantages of Benzimidazol fungicides, i.e., requiring correct timing and repeated applications and the problem of resistant strains. Calcium cyanamide has been shown to control foot rot diseases, leaf and ear diseases of small grains (Rieder 1981, supra).
Transgenic tobacco (Nicotiana tabacum) expressing the cah gene has been achieved (Maier-Greiner et al., Angew. Chem. Int. Ed. Engf. 30:1314-1315, 1991). The tobacco plants were infected with Agrobacterium tumefaciens containing a cah gene cassette, and the transformants were selected based on kanamycin resistance and regenerated. The transgenic tobacco plants were resistant to cyanamide and produced urea.
Zhou et al. (Plant Cell Reports 15:159-163, 1995) report the transformation of wheat using glyphosate-tolerant CP4 and GOX genes as a selectable marker. The CP4 and GOX genes were isolated from bacteria and confer resistance to glyphosate, which is the active ingredient in the non-selective herbicide Roundup (Monsanto Co.) The transformation efficiency for the CP4 and GOX genes was lower than that achieved using nptll (kanamycin resistance) or bar (glufosinate resistance) genes as selectable markers.
Ortiz et al. (Plant Cell Reports 15:877-881, 1996) report transformation of wheat using hygromycin resistance as a selectable marker. The hpt gene confers resistance to the antibiotic hygromycin. Ortiz et al. achieved an average transformation efficiency of 5.5% using the hpt gene, as compared to an efficiency of 2.6% for the bar-gene. The relatively high transformation efficiency was achieved in part due to the fact that the selection strategy did not attempt to eliminate all escapes. The escape rate was 5-10%.